Use of laccase in baking

ABSTRACT

A dough- or bread-improving composition comprising laccase as well as the use of the composition in the preparation of dough and baked products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National stage filing of PCT/DK94/00232 filed Jun.13, 1994, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a bread-improving or dough-improvingcomposition comprising an enzyme as well as to a method of preparing adough and/or a baked product by use of the composition.

BACKGROUND OF THE INVENTION

In the bread-making process it is known to add bread-improving and/ordough-improving additives to the bread dough, the action of which, interalia, results in improved texture, volume, flavour and freshness of thebread as well as improved machinability of the dough.

In recent years a number of enzymes have been used as dough and/or breadimproving agents, in particular enzymes which act on components presentin large amounts in the dough. Examples of such enzymes are found withinthe groups of amylases, proteases and cellulases, includingpentosanases.

Laccase (EC 1.10.3.2) is an enzyme catalyzing the conversion ofbenzenediols into benzosemiquinones according to the following formula:

4 benzenediol+O₂=4 benzosemiquinone+2H₂O.

The enzyme has been used in the paper and pulp industry as well as fordiagnosis, but the use of the enzyme in baking has never been suggested.

It is the object of the present invention to improve the properties ofdough and/or baked products by the use of a laccase enzyme.

BRIEF DISCLOSURE OF THE INVENTION

Accordingly, in a first aspect the present invention relates to abread-improving and/or a dough-improving composition comprising alaccase enzyme.

In the present context the terms “bread-improving composition” and“dough-improving composition” are intended to indicate compositionswhich, in addition to the enzyme component, may comprise othersubstances conventionally used in baking to improve the properties ofdough and/or baked products. Examples of such components are givenbelow.

Without being limited to any theory it is presently contemplated thatwhen a laccase enzyme is added to dough used for producing bakedproducts, it may exert an oxidizing effect on dough constituents andthereby serve to, e.g., improve the strength of gluten structures indough and/or baked products. Further improved properties may also beshown to be obtained.

In the present context the term “improved properties” is intended toindicate any property which may be improved by the action of a laccaseenzyme. In particular, the use of laccase results in an increased volumeand an improved crumb structure and softness of the baked product, aswell as an increased strength, stability and reduced stickiness andthereby improved machinability of the dough. The effect on the dough hasbeen found to be particularly good when a poor quality flour has beenused. The improved machinability is of particular importance inconnection with dough which is to be processed industrially.

The improved properties are evaluated by comparison with dough and/orbaked products prepared without addition of laccase in accordance withthe present invention.

In a second aspect, the present invention relates to a method ofpreparing a dough and/or a baked product prepared from the doughcomprising adding a laccase enzyme, optionally present in abread-improving or dough-improving composition, to the dough and/or toany ingredient of the dough and/or to any mixture of the doughingredients.

In further aspects, the present invention relates to a dough and a bakedproduct, respectively, produced by the present method as well as to apre-mix comprising a laccase enzyme. In the present context, the term“pre-mix” is intended to be understood in it's conventional meaning,i.e. as a mix of baking agents, normally including flour, which may beused not only in industrial bread-baking plants/facilities, but also inretail bakeries.

In a final aspect, the present invention relates to the use of a laccaseenzyme for the preparation of dough and/or baked products.

DETAILED DISCLOSURE OF THE INVENTION

While the laccase enzyme of the dough- and/or bread-improvingcomposition of the invention may be of any origin, including plantorigin, it is presently preferred that the laccase enzyme is ofmicrobial origin. Thus, a microbial enzyme is normally easier to produceon a large scale than a non-microbial enzyme of, e.g., plant origin.Furthermore, the microbial enzyme may normally be obtained in a higherpurity than enzymes of other origins, resulting in a lower amount ofundesirable enzymatic side-activities.

The microbial laccase enzyme may be derived from bacteria or fungi(including filamentous fungi and yeasts) and suitable examples include alaccase derivable from a strain of Aspergillus, Neurospora, e.g. N.crassa Podospora, Botrytis, Collybia, Fomes, Lentinus, Lentinus,Pleurotus, Trametes, Rhizoctonia, e.g. R. solani, Coprinus, e.g. C.plicatilis, Psatyrella, Myceliophtera, e.g. M. thermophila,Schytalidium, Polyporus, e.g. P. pinsitus, Phlebia, e.g. P. radita (WO92/01046), or Coriolus, e.g. C. hirsutus (JP 2-238885).

The laccase may be obtained from the microorganism in question by use ofany suitable technique. For instance, a laccase preparation may beobtained by fermentation of a microorganism and subsequent isolation ofa laccase containing preparation from the resulting fermented broth ormicroorganism by methods known in the art, but more preferably by use ofrecombinant DNA techniques as known in the art. Such method normallycomprises cultivation of a host cell transformed with a recombinant DNAvector capable of expressing and carrying a DNA sequence encoding thelaccase in question, in a culture medium under conditions permitting theexpression of the enzyme and recovering the enzyme from the culture.

The DNA sequence may be of genomic, CDNA or synthetic origin or anymixture of these, and may be isolated or synthesized in accordance withmethods known in the art.

The laccase enzyme to be included in the bread-improving and/ordough-improving composition of the invention may be in any form suitedfor the use in question, e.g. in the form of a dry powder or granulate,in particular a non-dusting granulate, a liquid, in particular astabilized liquid, or a protected enzyme. Granulates may be produced,e.g. as disclosed in U.S. Pat. No. 4,106,991 and U.S. Pat. No. 4,661,452(both to Novo Industri A/S), and may optionally be coated by methodsknown in the art. Liquid enzyme preparations may, for instance, bestabilized by adding nutritionally acceptable stabilizers such as asugar, a sugar alcohol or another polyol, lactic acid or another organicacid according to established methods. Protected enzymes may be preparedaccording to the method disclosed in EP 238,216.

Normally, for inclusion in pre-mixes or flour it is advantageous thatthe laccase enzyme preparation is in the form of a dry product, e.g. anon-dusting granulate, whereas for inclusion together with a liquid itis advantageously in a liquid form.

The bread- and/or dough-improving compostion of the invention mayfurther comprise another enzyme. Examples of other enzymes are acellulase, a hemicellulase, a pentosanase (useful for the partialhydrolysis of pentosans which increases the extensibility of the dough),a glucose oxidase (useful for strengthening the dough), a lipase (usefulfor the modification of lipids present in the dough or doughconstituents so as to soften the dough), a peroxidase (useful forimproving the dough consistency), a protease (useful for glutenweakening, in particular when using hard wheat flour), a peptidaseand/or an amylase, e.g. α-amylase (useful for providing sugarsfermentable by yeast).

The other enzyme components are preferably of microbial origin and maybe obtained by conventional techniques used in the art as mentionedabove. In a particular embodiment, the other enzyme component(s) may beproduced from the same source as the laccase in question and recoveredtogether with the laccase.

In addition or in an alternative to other enzyme components, thedough-improving and/or bread-improving composition may comprise aconventionally used baking agent, e.g. one or more of the followingconstituents:

A milk powder (providing crust colour), gluten (to improve the gasretention power of weak flours), an emulsifier (to improve doughextensibility and to some extent the consistency of the resultingbread), granulated fat (for dough softening and consistency of bread),an oxidant (added to strengthen the gluten structure; e.g. ascorbicacid, potassium bromate, potassium iodate or ammonium persulfate), anamino acid (e.g. cysteine), a sugar, and salt (e.g. sodium chloride,calcium acetate, sodium sulfate or calcium sulfate serving to make thedough firmer), flour or starch.

Examples of suitable emulsifiers are mono- or diglycerides, diacetyltartaric acid esters of mono- or diglycerides, sugar esters of fattyacids, polyglycerol esters of fatty acids, lactic acid esters ofmonoglycerides, acetic acid esters of monoglycerides, polyoxyethylenestearates, phospholipids and lecithin.

The bread-improving and/or dough improving composition of the inventionis typically included in the dough in an amount corresponding to 0.1-5%,such as 0.1-3% of the amount of flour to be added and typicallycomprises 0.5-30,000 Laccase Units per gramme.

In accordance with the method of the invention, in which laccase is usedfor the preparation of dough and/or baked products, the enzyme may beadded as such to the mixture from which the dough is made.Alternatively, the laccase enzyme may be added as a constituent of adough-improving and/or a bread-improving composition as described above.

The dosage of the laccase enzyme to be used in the method of the presentinvention should be adapted to the nature and composition of the doughin question. Normally, the enzyme preparation is added in an amountcorresponding to 5-100,000 Laccase Units. The Laccase Units (LACU) maybe determined by the assay described below in the Materials and Methodssection.

It is preferred that the laccase enzyme is added in an amountcorresponding to 50-20,000 LACU, such as 50-30,000 LACU, and inparticular 100-1500 LACU per kg of flour.

When one or more additional enzyme activities are to be added inaccordance with the method of the invention, these activities may beadded separately or together with the laccase preparation, optionally asconstituent(s) of the bread-improving and/or dough-improving compositionof the invention.

The other enzyme activities may be any of the above described enzymesand may be dosed in accordance with established baking practice.

As mentioned above the laccase enzyme is added to any mixture of doughingredients, to the dough, or to any of the ingredients to be includedin the dough, in other words the laccase enzyme may be added in any stepof the dough preparation and may be added in one, two or more steps,where appropriate. However, the enzyme should not be added together withany strong chemical or under conditions where the enzyme is inactivated.

The handling of the dough and/or baking is performed in any suitablemanner for the dough and/or baked product in question, typicallyincluding the steps of kneading the dough, subjecting the dough to oneor more proofing treatments, and baking the product under suitableconditions, i.e. at a suitable temperature and for a sufficient periodof time. For instance, the dough may be prepared by using a normalstraight dough process, a sour dough process, an overnight dough method,a low-temperature and long-time fermentation method, a frozen doughmethod, the Chorleywood Bread process, and the Sponge and Dough process.

The dough and/or baked product prepared by the method of the inventionare normally based on wheat meal or flour, optionally in combinationwith other types of meal or flour such as corn flour, rye meal, ryeflour, oat flour or meal, soy flour, sorghum meal or flour, or potatomeal or flour.

However, it is contemplated that the method of the present inventionwill function equally well in the preparation of dough and bakedproducts primarily based on other meals or flours, such as corn meal orflour, rye meal or flour, or any other types such as the types of mealor flour mentioned above.

In the present context the term “baked product” is intended to includeany product prepared from dough, either of a soft or a crisp character.Examples of baked products, whether of a white, light or dark type,which may advantageously be produced by the present invention are bread(in particular white, wholemeal or rye bread), typically in the form ofloaves or rolls, French baguette-type bread, pita bread, tacos, cakes,pancakes, biscuits, crisp bread and the like.

The dough of the invention may be of any of the types discussed above,and may be fresh or frozen.

From the above disclosure it will be apparent that the dough of theinvention is normally a leavened dough or a dough to be subjected toleavening. The dough may be leavened in various ways such as by addingsodium bicarbonate or the like or by adding a leaven (fermenting dough),but it is preferred to leaven the dough by adding a suitable yeastculture such as a culture of Saccharomyces cerevisiae (baker's yeast).Any of the commercially available S. cerevisiae strains may be employed.

As mentioned above, the present invention further relates to a pre-mix,e.g. in the form of a flour composition, for dough and or baked productsmade from dough, which pre-mix comprises a laccase enzyme. The pre-mixmay be prepared by mixing a laccase preparation or a bread-improvingand/or dough-improving composition of the invention comprising laccasewith a suitable carrier such as flour, starch, a sugar or a salt. Thepre-mix may contain other dough-improving and/or bread-improvingadditives, e.g. any of the additives, including enzymes, mentionedabove.

In a further aspect the invention relates to the use of a laccase enzymefor improving properties of a dough and/or a baked product madetherefrom. The type of laccase as well as the manner in which it may beused is described in detail above.

In a final aspect the invention relates to the use of laccase for thepreparation of pasta dough, preferably prepared from durum flour or aflour of comparable quality. The dough may be prepared by use ofconventional techniques and the laccase used in a similar dosage as thatdescribed above. The laccase is preferably of microbial origin, e.g. asdisclosed herein. It is contemplated that when used in the preparationof pasta the laccase results in a strengthening of the gluten structureand thus a reduction in the dough stickiness and an increased doughstrength.

MATERIALS AND METHODS

Enzymes

Laccase: A Rhizoctonia solani laccase produced by the Rhizoctonia solanistrain RS22 deposited with the International Mycological Institute,Genetic Resource Reference Collection, located at Bakeham Lane, EghamSurrey TW20 9TY on Sep. 3, 1993 under the terms of the Budapest Treatyand given the accession number IMI CC 358730. The Rhizoctonia solanilaccase is further described in co-pending application (U.S. Ser. No.08/172,331), the contents of which is hereby incorporated by reference.

Xylanase: A xylanase produced by the Humicola insolens strain DSM 1800available from the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH and further described in EP 507 723.

Pentosanase: Pentopan® available from Novo Nordisk A/S.

Fungamyl: A commercial fungal α-amylase preparation available from NovoNordisk A/S.

Determination of Laccase Activity

Laccase activity is determined by incubating a laccase containing samplewith syringaldazin (19 μM, Sigma No. S 7896) under aerobic conditions(30° C., 1 min., pH 5.5), whereby the syringaldazin is oxidized totetramethoxy azo bis-methylene quinone. The absorbance is measured at530 nm after 15 and 75 seconds and the difference between the measuredvalues is proportional with the laccase units per ml or grammes ofsample.

1 Laccase Unit (LACU) is the amount of enzyme which, under theprescribed reaction conditions, converts 1 μM syringaldazin per minute.

FAU—Fungal Alpha-Amylase Units

1 FA-unit (FAU) is the amount of enzyme which at 37° C. and pH 4.7breaks down 5260 mg of solid starch per hour.

FXU—xylanase Activity

The endo-xylanase activity is determined by an assay, in which thexylanase sample is incubated with a remazol-xylan substrate(4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R,Fluka), pH 6.0. The incubation is performed at 50° C. for 30 min. Thebackground of non-degraded dyed substrate is precipitated by ethanol.The remaining blue colour in the supernatant is determinedspectrophotometrically at 585 nm and is proportional to the endoxylanaseactivity.

The endoxylanase activity of the sample is determined relatively to anenzyme standard.

Preparation of Bread

Procedure

1. Dough mixing (Spiral mixer)

3 min. at 700 RPM

8 min. at 1400 RPM

the mixing time was determined and adjusted by a skilled baker so as toobtain an optimum dough consistence under the testing conditions used.

2. 1st proof: 30° C.-80% RH, 20 min.

3. Scaling and shaping;

4. Final proof: 32° C.-80% RH, 40 min.;

5. Baking: 225° C., 20 min. for rolls and 30 min for loaf.

Evaluation of Dough and Baked Products

Dough and baked products described in the examples were evaluated asfollows:

Loaf specific volume: the mean value of 4 loaves volume are measuredusing the traditional rape seed method. The specific volume iscalculated as volume ml per g bread. The specific volume of the control(without enzyme) is defined as 100. The relative specific volume indexis calculated as:${{Specific}\quad {{vol}.\quad {index}}} = {\frac{{specific}\quad {{vol}.\quad {of}}\quad 4\quad {loaves}}{{{spec}.\quad {vol}.\quad {of}}\quad 4\quad {control}\quad {loaves}} - {*100}}$

The dough stickiness and crumb structure are evaluated visuallyaccording to the following scale:

Dough stickiness: almost liquid 1 too sticky 2 sticky 3 normal 4 dry 5Crumb structure: very poor 1 poor 2 non-uniform 3 uniform/good 4 verygood 5

Shock test: After the second proof a pan containing the dough is droppedfrom a height of 20 cm. The dough is baked and the volume of theresulting bread is determined.

EXAMPLES Example 1

Wheat flour pan bread Recipe Wheat flour 100% Yeast 4% Salt 1.5% Sugar1.5% Water 59%

Flour type: Pastetli-Intermid without ascorbic acid (Intermill,Schweiz).

TABLE 1 Laccase LACU/kg  0 600 1000  flour Specific volume 100 100 107index After shock Specific volume 100 109 106 index Crust crispy normalimproved improved

Laccase has a significant effect on improving the baking quality offlour without oxidant. The dough stability is improved by the additionof laccase, because the bread with laccase regain more volume than thecontrol after the dough have been shocked.

Example 2

Whole meal bread Recipe Wholemeal flour (Intermill-Graham) 100%  Yeast4% Salt 2% Malt 1% Gluten 3% Water 75% 

TABLE 2 Laccase LACU/kg flour 0 500 1000 1000 1000 0 Pentopan g/kg flour0 0.15 Fungamyl FAU/kg flour 15 15 Specific volume index 100 101 101 105108 110 after shock, Specific 100 105 108 106 109 105 volume index

For the whole meal bread, addition of laccase reduces dough stickinessduring dough handling. Furthermore, the dough becomes more stable (asshown by the fact that bread treated with laccase can regain more volumecompared to the control after shock test).

Example 3

Bread Prepared with Wheat Flour Containing Ascorbic Acid

The wheat flour used was Manitaba flour, a Danish baking flour withascorbic acid (avaible from Valsemøllerne A/S, Denmark).

TABLE 3 Laccase LACU/kg fl. 0 1000 1000 1000 1000 Pentopan g/kg fl. 00.15 0.15 Xylanase FXU/kg fl. 0 50 50 Fungamyl FAU/kg fl. 0 30 30 Doughstickiness 4 4.5 4 3.5 4 3.5 4.5 3 Sp. vol-.ind 100 104 116 116 117 117112 111 Crumb structure 3 4 4 4 4 4 4 3.5 Dough firmness 4 4 4 3.5 4.53.5 4 3.5

Addition of laccase reduces the dough stickiness. Enzymes such aspentosanase/xylanase and fungal amylase can cause dough stickiness anddough softness. When combining laccase with one of these enzymes thedough became normal. This is desirable for baking processes in whichdough stickiness is not acceptable.

Example 4

Flour

Whole wheat flour (Intermill, Schweiz)

Prima flour (Havnemøllerne A/St Denmark),

Reform flour (Havnemøllerne A/S, Denmark)

Manitoba flour (Valsemøllerne A/S, Denmark),

Pasteli-flour (Intermill, Schweiz).

Procedure Flour 300 g NaCl  6 g

Laccase was used in the amount indicated in the tables.

Water was added in an amount sufficient to give a consistency of500+/−20 FU in Brabender Farinograph.

Extensograph measurements were conducted 45, 90, and 135 min,respectively, of dough resting. The extensograph used was fromBranbender and Duisburg, Germany, and was used as recommended by themanufacturer.

The results obtained appear from the following tables. It is evidentthat the use of laccase increases the dough resistance to extension ofdough prepared from different types of flour. The resistance toextension increases with increasing dosages of laccase.

TABLE 4 WHOLE WHEAT FLOUR PROV- RESISTANCE EXTEN- LACCASE ING to SIBIL-MAXI- (U/kg TIME ENERGY EXTENSION ITY MUM FLOUR) (min) (cm2) (BU) (mm)(BU) 0 45 56 273 135 273 600 64 333 125 338 1000 64 343 121 348 1500 66371 117 375 0 90 61 361 114 363 600 74 483 106 487 1000 76 502 107 5161500 75 534 99 543 0 135 65 393 115 397 600 77 543 101 554 1000 79 57299 581 1500 73 569 94 573

TABLE 5 REFORM FLOUR Proving Time: 45 min RESISTANCE LACCASE to MAXI-(U/kg ENERGY EXTENSION EXTENSIBILITY MUM FLOUR) (cm2) (BU) (mm) (BU) 0110 494 131 609 600 111 508 128 650 1000 120 562 127 704 1500 112 564122 669

TABLE 6 “PASTETLI” FLOUR PROV- RESISTANCE EXTEN- LACCASE ING to SIBIL-MAXI- (U/kg TIME ENERGY EXTENSION ITY MUM FLOUR) (min) (cm2) (BU) (mm)(BU) 0 45 86 342 145 403 600 101 385 149 480 1000 101 394 146 492 1500106 473 133 573 0 90 103 437 144 521 600 114 535 131 631 1000 120 617125 705 1500 124 758 112 842 0 135 109 499 134 589 600 116 636 118 7301000 115 707 111 787 1500 121 842 102 906

TABLE 7 MANITOBA Proving Time: 45 min RESISTANCE LACCASE to MAXI- (U/kgENERGY EXTENSION EXTENSIBILITY MUM FLOUR) (cm2) (BU) (mm) (BU) 0 119 421149 597 600 133 473 148 681 1000 135 519 144 708 1500 135 567 136 748

TABLE 8 PRIMA FLOUR Proving Time: 45 min RESISTANCE LACCASE to MAXI-(U/kg ENERGY EXTENSION EXTENSIBILITY MUM FLOUR) (cm2) (BU) (mm) (BU) 0121 496 141 629 600 115 528 127 678 1000 126 565 132 711

What is claimed is:
 1. A dough composition suitable for baking, comprising flour, water, and a laccase enzyme in an amount effective to increase resistance of the dough composition to extension.
 2. The dough composition of claim 1, wherein the laccase enzyme is derived from Rhizoctonia, Coprinus, Myceliophtera, Polyporous, Coriolus, or Phlebia.
 3. The dough composition of claim 1, wherein the composition further comprise an enzyme selected from the group consisting of cellulase, a hemicellulase, a pentosanase, a glucose oxidase, a lipase, a peroxidase, a protease, a peptidase or an amylase.
 4. The dough composition of claim 1, wherein the laccase enzyme is added in amount of from 5 to 100,000 Laccase Units per kg of flour.
 5. The dough composition of claim 1, wherein the laccase enzyme is added in amount of from 50 to 30,000 Laccase Units per kg of flour.
 6. The dough composition of claim 1, wherein the laccase enzyme is added in amount of from 50 to 20,000 Laccase Units per kg of flour.
 7. The dough composition of claim 1, wherein the laccase enzyme is added in amount of from 100 to 1500 Laccase Units per kg of flour.
 8. A method of preparing a dough composition, comprising adding to a dough composition a laccase enzyme in an amount effective to increase resistance of the dough composition to extension.
 9. The method of claim 8, wherein the laccase enzyme is derived from Rhizoctonia, Coprinus, Myceliophtera, Polyporous, Coriolus, or Phlebia.
 10. The method of claim 8, further comprising adding to the dough composition an enzyme selected from the group consisting of cellulase, a hemicellulase, a pentosanase, a glucose oxidase, a lipase, a peroxidase, a protease, a peptidase or an amylase.
 11. The method of claim 8, wherein the laccase enzyme is added in amount of from 5 to 100,000 Laccase Units per kg of flour.
 12. The method of claim 8, wherein the laccase enzyme is added in amount of from 50 to 30,000 Laccase Units per kg of flour.
 13. The method of claim 8, wherein the laccase enzyme is added in amount of from 50 to 20,000 Laccase Units per kg of flour.
 14. The method of claim 8, wherein the laccase enzyme is added in amount of from 100-1500 Laccase Units per kg of flour.
 15. A method of preparing a bread, comprising: a) providing a dough composition; b) adding to the dough composition a laccase enzyme in an amount effective to increase resistance of the dough composition to extension; and c) baking the dough composition. 